Fluorescein esters and ethers and the preparation thereof

ABSTRACT

Fluorescein esters and ethers including the dioleyl derivative of fluorescein are disclosed. These compounds have the useful property of being fluorescent under the influence of the proper frequency of electromagnetic irradiation. Further, dioleyl fluorescein is readily incorporated into low-density lipoproteins which can then become part of the cell matter; the compounds retain their fluorescent properties during this procedure. The fluorescence allows cells to be identified and separated. The fluorescein esters and ethers are prepared from fluorescein and the novel reagent O-alkenyl-N,N&#39;-dialkylisourea.

BACKGROUND OF THE INVENTION

Fluorescein is a known fluorescent agent, that is in the presence ofultraviolet light, solutions of the compound fluoresce, or give offlight. This characteristic has been used to examine subterranian watersfor sources of underground springs, and detecting sources ofcontamination of drinking water and infiltration with waste waters offactories. An extension of this use would be to have fluoresceinincorporated into biological systems, such as cells to follow the pathsof such cells, or to separate certain cells from other cells. However,this has not been possible owing to the non-incorporation ornon-selective incorporation of fluorescein into such biological systems.The present invention solves this problem by providing means forincorporating fluorescein into cells, retaining the fluorescentquantities of the compound.

SUMMARY OF THE INVENTION

This invention concerns the ester and ether derivatives of fluoresceinand procedures for preparing such compound from fluorescein andO-alkenyl-N,N'-dialkylisourea. Thus, it is an object of this inventionto provide for such fluorescein derivatives. A further object is todescribe the use of such fluorescein derivatives as fluorescent tracersto detect the presence of biological cells. A still further object is toprovide for the preparation of such fluorescein derivatives. A stillfurther object is to provide for the novel reagent used in thepreparation of such fluorescein derivatives. Further objects will becomeapparent from a reading of the following description.

DESCRIPTION OF THE INVENTION

Fluorescein has the following structure: ##STR1## The compound of thisinvention, fluorescein esters and ethers have the following structure(I): ##STR2## wherein R is an alkenyl group of from 12 to 30 carbonatoms and from 1 to 4 double bonds.

Examples of such alkenyl groups are lauroleyl ((Z)-9-dodecene),myristoleyl ((Z)-9-tetradecene), palmitoleyl ((Z)-9-hexadecene), oleyl((Z)-9-octadecene), triacontyl ((Z)-9-triacontene), linoleyl((Z,Z)-9,12-octadecadiene) linolenyl ((Z,Z,Z)-9,12,15-octadecatriene),eleostearyl (Z,Z,Z)-9,11,13-octadecatriene), arachidonyl((Z,Z,Z,Z)-5,8,11,14-eicosatetraenoic), and the like.

The preferred compounds of this invention are realized when the doublebonds are all in a cis-configuration. Further preferred compounds arerealized when there is one double bond in the alkenyl group. Stillfurther preferred compounds are realized when the alkenyl group containsfrom 16 to 20 carbon atoms. The most preferred alkyl group contains 18carbon atoms and is oleyl ((Z)-9-octadecene)

These compounds are prepared by reacting fluorescein with a reagentcapable of alkylating the carboxylic acid as well as the phenolichydroxyl groups. Common alkylating reagents known in the art foralkylating carboxylic acids and phenols were tried and found to beunsatisfactory. The novel reagent O-alkenyl-N,N'-dialkylisourea (II) wasthen prepared and this compound was found to successfully andsimultaneously alkylate both the carboxylic acid and phenolic hydroxylsof fluorescein. The structure of this reagent is: ##STR3## wherein R isas defined above and R₁ and R₂ are independently loweralkyl of from 1 to6 carbon atoms or cycloalkyl of from 4 to 6 carbon atoms. The loweralkylgroups may be either straight or branched chain. The preferred compoundsare those wherein R₁ and R₂ are the same. Further preferred compoundsare when R₁ and R₂ are isopropyl or cyclohexyl. The most preferredcompounds is when R₁ and R₂ are isopropyl.

The novel reagent of this invention (II) is prepared by reacting anN,N'-dialkyl-carbodiimide (III) with an alcohol (IV) as in the followingreaction scheme: ##STR4## wherein R, R₁, and R₂ are as defined above.

Both starting materials are known in the art. In the reaction to preparecompound (II), the two compounds are combined in the presence of acuprous halide (CuX). The cuprous halide, preferably cuprous chloride,is present in catalytic amounts. The reaction may be carried out in aninert aprotic solvent such as benzene, toluene, halogenated hydrocarbonsand the like, however, it is preferred to combine the reactants withoutany solvent. The reaction is generally carried out using equimolaramounts of compounds III and IV, however, excesses of one reactant overthe other has been found to be neither detrimental nor beneficial.Equimolar amounts are preferred, since this avoids removal of the excessreactant. The reaction is carried out at from room temperature to about100° C. and is generally complete in from 1/2 to 24 hours. It ispreferred to carry out the reaction at room temperature wherein thereaction has been found to be generally complete in about 3-5 hours. Theproduct (II) is recovered from the reaction mixture using techniquesknown to those skilled in the art.

The products of this invention, ester and ether derivatives offluorescein (I) are prepared by reacting fluorescein with the aboveprepared O-alkenyl-N,N'-dialkylisourea (II). Again, generally no solventis used, or an inert solvent may be employed. Preferred solvents aredimethyl formamide, benzene, toluene, halogenated hydrocarbons, and thelike. It is most preferred to carry out the reaction indimethylformamide. The reaction is carried out at an elevatedtemperature of from 70°-200° C. Preferably from 90°-110° C. If a solventis used with a boiling point less than the desired reaction temperature,a pressure bomb should be employed. The reaction is generally completein from 2-48 hours. At the preferred temperature range, the reaction isgenerally complete in 5-18 hours. The reactants are generally used insubstantially equimolar amounts or preferably with a slight excess ofthe O-alkenyl-N,N'-dialkylisourea.

The reaction has been found to alkenylate the carboxylic acid group andthe phenolic hydroxyl group simultaneously and with equal facility underthe above reaction conditions. This reaction may also be used toalkenylate other carboxylic acid groups and phenolic hydroxyl groupsfound on other biologically active molecules and, with the alkenylfunction thereon, can be incorporated into the cells of biologicalsystems.

The dialkenyl fluorescein finds utility in providing for the readyvisualization of cells in biological systems and fluids.

The plasma lipoproteins are a family of globular particles each of whichconsists of a core of neutral lipid (primarily triglyceride orcholesteryl ester) surrounded by a coat of phospholipid and protein. Onefraction of the plasma lipoprotein has been separated therefrom and isknown as low-density lipoprotein (LDL). The LDL is the major cholesterolcarrying lipoprotein of human plasma. The LDL attaches to theappropriate binding site of a cell and is drawn within the cell. The LDLcoat is then ruptured and the contents of the core incorporated withinthe cell. Recently a method has been described by which the core of LDLis removed by extraction with heptane and replaced by exogenouscholesteryl esters. The LDL has retained its affinity for the cellbinding site after this procedure and the exogenous cholesteryl estershave been incorporated into the cells.

It is thus realized that perhaps other exogenous materials could beplaced into the LDL core to be eventually incorporated into a cell. Themain barrier to this procedure is the requirement that the material tobe placed in the LDL core be highly lipophilic.

In cell research, it is highly desirable to separate specific cells froma mixture of cells.

The instant procedure provides that cells may be visualized. Once cellsare visualized, they may then be separated using techniques known tothose skilled in the art.

Fluorescein is a highly fluorescent molecule and would readily aid inthe identification and separation of cells, if it could be incorporatedtherein without affecting the cell. Unfortunately fluorescein is not atall lipophylic and, thus, cannot be incorporated into the cell via theLDL core transfer procedure. It has been found, however, that thealkenyl derivatives, particularly the dioleyl derivative of fluoresceinis sufficiently lipophilic to be incorporated into the LDL core. Inaddition, the LDL with the dioleyl fluorescein core retains its affinityfor the cell binding site and further, the cell, once it has rupturedthe LDL coat and incorporated the dioleyl fluorescein therein, is highlyfluorescent.

The fluorescent cells are, thus, easily visualized under visible orultraviolet light and fractions of cells thus are readily separated.Generally, this is accomplished with a fluorescence activated cellsorter. In addition, it has been found that the fluorescent natureimparted to the cells does not affect the properties of such cells suchthat they may be used for whatever purposes the researcher intendswithout untoward effects.

The foregoing technique thus provides for an easily detected visualprobe that can be used to determine whether or not a cell in a cultureexpresses LDL receptors. The use of fluorescent LDL also enhances theease with which cells are screened for mutations in the LDL uptakepathway, including mutagens in patients with familialhypercholesterolemia as well as mutations that are created through invitro mutagenesis in cultured cells.

The following examples are presented in order that the invention mightbe more fully understood. They are not to be construed as limitative ofthe invention.

EXAMPLE 1 O-Oleyl-N,N'-Diisopropylisourea

A mixture of N,N'-diisopropylcarbodiimide (12.6 g, 0.1 mole), oleylalcohol (26.8 g, 0.1 mole) and cuprous chloride (60 mg) is stirred atroom temperature for 4 hours. Infrared analysis shows the reaction to bevirtually complete. The reaction mixture is placed on a column ofalumina (454 g) and eluted with methylene chloride to give 36.2 g ofO-oleyl-N,N'-diisopropylisourea. The product is identified by massspectrometry (molecular ion at m/e 394), infrared spectroscopy (the C═Nband at 1660 cm⁻¹) and nuclear magnetic resonance in deuteratedchloroform; resonances are given in δ relative to tetramethylsilane:0.9-2.2 (mutiplet 37 protons); 2.9-3.8 (multiplet 3 protons); 4.0(triplet, 2 protons) 5.3 (multiplet, 2 protons).

EXAMPLE 2 Dioleyl Fluorescein

A mixture of fluorescein (1.33 g, 4.0 mmole) andO-oleyl-N,N'-diisopropylisourea (3.48 g, 8.8 mmole) are heated under ablanket of nitrogen for 16 hours at 142° C. Analysis of an aliquot on athin layer chromatography plate (silica gel eluting with 95% chloroform5% methanol) indicates that all of the fluorescein is reacted. Theresidue is taken up in 100 ml. of ether, filtered and passed through 2.0g of silica gel to give 3.91 g of crude material. The crude material ispurified by preparative high pressure liquid chromatography using 325 gof silica gel on a Waters Associates System 500 instrument, at a flowrate of 200 ml per minute of a 4:1 mixture of mthylene chloride andether. The product, dioleyl fluorescein, separates with an Rf of 0.35.The product is identified by mass spectrometry (field desorption) with asingle peak at M+H of 834; infrared spectroscopy (carbonyl absorption at1720 cm⁻¹ ; nuclear magnetic resonance in deuterated chloroform;resonances are given in δ relative to tetramethylsilane: 0.9-2.2(multiplet, 62 protons) 6.01 multiplet, 4 protons), 5.3 (multiplet, 4protons) 6.4-8.3 (multiplet, 10 protons).

EXAMPLE 3

Following the procedure of Example 1 using N,N'-diisopropylcarbodiimide,cuprous chloride and the following alcohols:

Lauroleyl alcohol

Myristoleyl alcohol

Palmitoleyl alcohol

Triacontyl alcohol

Linoleyl alcohol

Linolenyl alcohol

Eleostearyl alcohol

Arachidonyl alcohol

The following compounds are produced:

O-Lauroleyl-N,N'-diisopropylisourea

O-Myristoleyl-N,N'-diisopropylisourea

O-Palmitoleyl-N,N'-diisopropylisourea

O-Triacontyl-N,N'-diisopropylisourea

O-Linoleyl-N,N'-diisopropylisourea

O-Linolenyl-N,N'-diisopropylisourea

O-Eleostearyl-N,N'-diisopropylisourea

O-Arachidonyl-N,N'-diisopropylisourea

EXAMPLE 4

Following the procedure of Example 2, using fluorescein and thecompounds prepared in Example 3, the following compounds are prepared:

Dilauroleyl fluorescein

Dimyristoleyl fluorescein

Dipalmitleyl fluorescein

Ditriacontyl fluorescein

Dilinoleyl fluorescein

Dilinolenyl fluorescein

Dieleostearyl fluorescein

Diarachidonyl fluorescein

What is claimed is:
 1. A compound having the formula: ##STR5## wherein Ris an alkenyl group of from 12 to 30 carbon atoms with from 1 to 4 cisdouble bonds.
 2. The compound of claim 1 wherein the R group containsone double bond.
 3. The compound of claim 2 wherein the R group containsfrom 16 to 20 carbon atoms.
 4. The compound of claim 3 wherein the Rgroup contains 18 carbon atoms.
 5. The compound of claim 4 which isdioleyl fluorescein.
 6. A process for the preparation of the compound ofclaim 1 which comprises reacting fluorescein at from 70° to 200° C. forfrom 2 to 48 hours either without a solvent or in the presence of aninert solvent, with a compound having the formula: ##STR6## wherein R isdefined in claim 1 and R₁ and R₂ are independently loweralky of from 1to 6 carbon atoms or cycloalkyl of from 4 to 6 carbon atoms.